The research proposed will be a systematic biochemical investigation of the mechanism and regulation of E. coli. RNA Polymerase. Several gene regulatory systems will be investigated in vitro. The focus throughout will be to understand how DNA sequences in the control regions of genes contribute to the regulation of transcription initiation. The mechanism of action of several transcriptional activators and repressors will be examined. The effects of DNA supercoiling on selected systems will also be investigated. In two bacteriophage systems, the effects of convergent transcription between two (or more) opposing promoters will be studied. The experiments proposed rely on standard biochemical assays for determining in vitro promoter strength. These results will be complemented in some cases with direct measurements of in vivo RNA chain initiation frequency and in other cases with direct measurements of in vitro binding of RNA polymerase and other regulatory proteins. The answers to the questions we have posed will contribute to the biochemical description of the control of gene expression.